Transposon in cloning DNA

Abstract

Processes and new genetic materials are provided for cloning specific DNA fragments by using a unique conjugative transposon designated Tn916. The transposon is used to first target specific genes by insertional inactivation. A restriction fragment containing the inserted transposon is then inserted into a plasmid vector and transformed into Escherichia coli or other suitable host by selection for the transposon encoded tetracycline (Tc) resistance. The transformants so produced are then grown in the absence of tetracycline conditions under which Tn916 excises from the chimeric plasmid thus restoring the integrity of the DNA into which the transposon was originally inserted. This process provides a new and useful way of producing new life forms that are useful for making desired products having established utility.

We claim: 
     
       1. A process for cloning a gene which comprises, (1) introducing excising transposon Tn 916 by transformation, conjugation, or transduction into a host microorganism which comprises a gene which is to be cloned,   (2) selecting for the transformed microorganisms which comprise Tn 916 inserted into said gene as evidenced by the insertional inactivation of the phenotype encoded for by said gene,   (3) isolating the DNA which contains Tn 916 from said selected transformed microorganisms,   (4) cleaving said isolated DNA with a suitable restriction enzyme so as to leave the Tn 916 and the gene intact,   (5) ligating said cleavage DNA fragment which comprises the Tn 916 and the cloned gene to a vector,   (6) introducing the chimaeric DNA produced in step (5) into a host microorganism by transformation,   (7) selecting for the transformed microorganisms which comprise said chimaeric DNA by screening for tetracycline resistance, and   (8) culturing said selected transformed microorganism in the absence of tetracycline so as to excise Tn 916 from said chimaeric DNA and thereby restore the phenotypical trait encoded for by the cloned gene.   
     
     
       2. A process for cloning a gene which comprises, (1) introducing excising transposon Tn 916 by transformation, conjugation, or transduction into a bacteria which comprises a gene which is to be cloned,   (2) selecting for the transformed bacteria which comprise Tn 916 inserted into said gene as evidenced by the insertional inactivation of the phenotype encoded for by said gene,   (3) isolating the DNA which contains Tn 916 from said selected transformed bacteria,   (4) cleaving said isolated DNA with a suitable restriction enzyme so as to leave the Tn 916 and the gene intact,   (5) ligating said cleavage DNA fragment which comprises the Tn 916 and the cloned gene to a vector,   (6) introducing the chimaeric DNA produced in step (5) into a bacteria by transformation,   (7) selecting for the transformed bacteria which comprise said chimaeric DNA by screening for tetracycline resistance, and   (8) culturing said selected transformed bacteria in the absence of tetracycline so as to excise Tn 916 from said chimaeric DNA and thereby restore the phenotypical trait encoded for by the cloned gene.   
     
     
       3. The process of claim 2 wherein the bacteria is the genus Bacillus. 
     
     
       4. The process of claim 2 wherein the bacteria is the genus Streptococcus. 
     
     
       5. The process of claim 2 wherein the bacteria is the genus Streptomyces. 
     
     
       6. The process of claim 2 wherein the species is Streptococcus mutans. 
     
     
       7. The process of claim 2 wherein the species is Streptococcus faecalis. 
     
     
       8. The process of claim 2 wherein the bacteria is Streptococcus faecalis, the restriction enzyme is EcoR1, the plasmid vector is PGL101, and the host is E. coli.